The structure and function of creatine kinase, particularly in the ligand state has been studied by the use of environmentally sensitive spectral probe. The enzyme can be labeled with 2 equivalents of 2-chloromercuri-4-nitrophenol without the loss of catalytic efficiency. The bound spectral probe can be utilized as a remarkably effective and exquisitely sensitive indicator of a variety of substrate-induced, presumably localized conformational changes in the enzyme. For instance, although the stoichiometry of the reaction and the enzymatic activity were unaffected, the rates of reaction of 2 eq of chromophore with many different enzyme-ligand complexes varied enormously. Moreover, the binding of ligands, singly or in various combinations, to the labeled enzyme elicited diverse spectral changes of the bound nitrophenol. In a series of complexes that ultimately make up the transition state analog: E, E-MgADP, E-MgADP-nitrate or E-MgADP-creatine, and E-MgADP-nitrate-creatine, the bound nitrophenol spectral changes differed distinctly for each complex, and the rates of reaction of the mercurial decreased markedly in the order: 0, 2-, 3-or 13, and 200-fold. Both the differences in the rates of reaction of the chromophore with various enzyme-ligand complexes and the varied spectral perturbations of the enzyme-bound reporter are distinctive manifestations of the cooperative substrates binding effects and are indicative of a graded degree of substrate-induced conformational changes.